Selection and isolation of microfungi for use in biological control of parasitic nematodes in animals

ABSTRACT

In a process for the selection and isolation of predacious fungi for use in the biological control of parasitic nematodes in animals, compost and soil samples are subjected to a multi-step process consisting of 1) a primary stress selection step done in vitro; 2) a secondary stress selection step done in vivo, wherein the fungi which survived the in vitro stress selection step, are selected on the basis of their ability to pass through living animals and 3) isolation of the predacious fungi passing both selection steps and exhibiting a predacious capacity of at least 50% by dung pat assay. These fungi can than be subject to an optional field test experiment. The selected predacious fungi can be administered to the animals as a feed additive or in the form of a device, such as a lick stone or bolus. The selected fungi can also be used in a composition for veterinary treatment.

This application was filed under 35 USA 371 as the national phase ofPCT/DK92100269.

This application was filed under 35 USA 371 as the national phase ofPCT/DK92100269.

FIELD OF THE INVENTION

The present invention relates to a process for selection and isolationof nematophagous fungi to be used as biological control agents againstinfective larvae of parasitic nematodes such as Ostertagia ostertagi andCooperia oncophora (Trichostrongylidae).

BACKGROUND OF THE INVENTION

Young calves which are put to pasture for the first time are susceptibleto a large number of germs, i.e. threadlike small parasitic nematodes(Trichostrongylidae), which are parasites in the abomasum and intestineof cattle. These infections can cause serious diseases, such asdiarrhea, indigestion, loss of weight, and death. But it is inparticular the relatively mild infections which are of economicimportance, since they are very widespread and result in poor growth, inparticular at the end of the season.

Infected calves secrete the very small eggs of the nematodes in theirfaeces. FIG. 1 shows the life cycle of cattle nematode parasites. Themature parasites multiply by laying eggs in the intestinal tract of thecattle. The eggs have a size of 0.05-0.1 mm and are thin shelled. Theyare excreted with the dung of the animals. The larvae develop from theeggs in the cow pats on the pasture. The larvae go through three larvalstages (L₁, L₂ and L₃). The third larval stage (L₃), which is infective,retain the cuticle of the second larval stage (L₂) as a protectivesheath until they enter their host. In the cow pat, temperature,moisture, oxygen pressure and the natural enemies of the parasites aredecisive for the number of eggs that develop to the infective stage. Theinfective larvae, having a length of up to 1 mm, are spread from the cowpat to the surrounding grass, in particular in connection with rainyweather. Most of the larvae are present within 30 cm from the rim of thecow pats. On the grass they may be eaten by grazing animals, and thenthey can continue their development in the intestinal tract of theanimals up to maturity.

The parasitic development starts when L₃ larvae are ingested by cattle.L₃ exsheath in the rumen. A few days after the infection, exsheathed L₃enter the abomasal glands, where they differentiate and increase insize. Three stages occur (L₃, L₄ and L₅), separated by two moults.Immature adult parasites (L₅) emerge from the glands around 18 daysafter the infection. During the next few days, they become sexuallymature on the surface of the abomasal mucosa, resulting in excretion ofeggs.

Since nematode parasites often cause great losses to farmers, it is verymuch of interest to control these parasites. Traditionally, this takesplace by medical treatment, but farmers also try in various ways toprotect grazing animals against eating large amount of larvae, e.g. byreducing the density of animals on the pasture so that they are notforced to graze the highly infected areas close to their own excrements.Animals can also be moved to a clean or low-infected field at the end ofthe summer when the danger of infection usually increases strongly.

However, these measures have a number of economic and practicaldrawbacks, making it desirable to find alternative methods of control.Thus, for a number of years efforts have been devoted to the developmentof methods for biological control of larvae of nematodes in cow patsalready before they are spread as infective larvae to the grass.Biological control comprises the use of natural enemies of thenematodes, which are nematophagous fungi, also called predacious fungi,in this case.

Predacious fungi are microfungi which do not develop fruiting bodies, asis known from e.g. mushrooms, but have a growth form resembling mold.Predacious fungi are special having developed organs that are able tocapture and kill small nematodes, including infective larvae ofnematodes. Predacious fungi are originally terricolous fungi, but it hasbeen found that they can also grow in cow pats. It is precisely heretheir beneficial effect should be used to kill large numbers of theinfective larvae of parasitic intestinal worms.

FIG. 2 shows a nematode larva captured by a predatory fungus. The larvais captured in a sticky net consisting of three strong arcs. Where thereis contact, the parasite is penetrated. Fungal hyphae extend from hereto grow out and fill the body of the nematode which is eventuallykilled. Then the inner organs are dissolved and absorbed. The fungalmycelium inside the parasite is shown by dotted lines.

To utilize predacious fungi in practice it is necessary to select fungalspecies which can pass through the intestinal tract of cattle alive.Moreover, methods should be developed to produce large amounts of fungalmaterial. It will hereby be possible to place predatory fungi in cowpats in the period during the grazing season when many parasite nematodelarvae develop in cow pats.

So far all attempts to isolate efficient fungi which are able to passthe intestinal tract of animals, and thereby usable in biologicalcontrol, have failed. It has also not been possible by any means to coatthe fungal material in order to protect said material against exposureto the enzymes in the intestinal tract.

Since the discovery of nematophagous fungi about one hundred years ago,several attempts have been made at using these organisms as biologicalcontrol agents against parasitic nematodes causing diseases in plantsand animals. The nematophagous fungi capable of destroying free livingnematodes can be divided into two groups, predaclous and endoparasitic(G. L. Barron; The nematode-destroying fungi; Topics in Mycobiology No.1, Canadian Biological Publications Ltd., Canada (1977)). J. Gronvold etal., (1987), Journal of Helminthology, 61:65-71 (1987); ibid.,62:271-280 (1988); ibid., 63:115-126 (1989), have shown that thepredacious fungus Arthrobotrys oligospora is capable of reducing thenumber of infective larvae (L₃) of the bovine trichostrongyles Cooperiaoncophora and Ostertegia ostertagi in dung and herbage. Arthrobotrysoligospora is one of the species most investigated in attempts ofbio-control (J. Gronvold et al., Journal of Helminthology, 69;119-125(1985); P. Nansen et al., Veterinary Parasltology, 26:329-337 (1988)),but it is not necessarily the most efficient one.

All previous attempts have failed because of the lack of nematophagousfungi, Including Arthrobotrys oligospora, which are able to pass throughthe intestinal tract of the animal and subsequently reduce the Infectionof the herbage effectively.

M. Peloille, IOBC/WPRS Bulletin XIV/2 (1991) has studied the criteriawhich should be taken into consideration when selecting effectivepredacious fungi. These criteria are growth rate at differenttemperatures, predacious activity, ability to survive passage throughthe intestinal tract of animals, end ability to produce chlamydosporeswhich can be used for the dispersal of the fungi. Duddingtonia flagransis mentioned as the species which fulfills all the requirements best.The conclusion which is not based upon scientifically correctexperiments is very misleading, because only few of the Duddingtoniafungi actually survive the passage through the Intestinal tract ofruminants.

One reason that the experiments are not scientifically correct is thatit is not documented that the experimental and the control animals werecomparable with respect to the number of parasite eggs. Furthermore, theanimals have only been fed once with a very large dose of the predatoryfungus (500 g), end even though this passes through the intestinal tractof the animals within 48 hours, the predatory fungus is claimed to beeffective for up to 96 hours. Furthermore, there is no reference sampletaken at the beginning of the experiment, since the results simply beginafter 24 hours. It is. also Important to mention that the author basesher conclusion solely on pure laboratory experiments. It is notdocumented that the effect of the fungus Is maintained in fieldexperiments.

These defects ere remedied by the selection technique according to theinvention, which will be described more fully below.

SUMMARY OF THE INVENTION

An object of the present invention is therefore to provide a methodenabling isolation of predacious fungi which have the unusual ability togrow in the dung of the calves after having passed the intestinal tractof the animals. In other words, it has now become possible to feedcalves with predacious fungi, which results in a substantial reductionin the content of infective larvae in the cow pats and the subsequentspreading of infective larvae to the surrounding herbage.

Surprisingly it has thus been found that it is possible to select andisolate specific predacious fungi which are effective biological controlagents when administered to cattle, sheep, goats, pigs and horses. Thepresent fungi are capable of surviving in the faeces of the animals toan extent such that the number of parasites is reduced by at least 50%.Two of the most preferred fungi which can be selected and isolated bythe method of the invention, are both strains of the speciesDuddingtonia flagrans, and they have been found capable of reducing theinfection level of the infective larvae spread from cow pats to thegrass by approximately 70-90%.

Accordingly, the invention concerns a process for the selection andisolation of nematophagous fungi for use in biological control whereincompost and soil samples are subjected to a three-step selectionprocedure and subsequently spread on tetracycline chloride/water agar(TCC-WA) plates. The three-step selection procedure consists of

1) a primary stress selection step in vitro wherein the samples areexposed to diluted rumen fluid at 39° C. for 24 hours, and a furtherselection step in vitro wherein the fungi surviving the primary stressselection step are tested in pure culture by exposure to a range offluids resembling those of the alimentary tract of domestic animals, and

2) a secondary stress selection step in vivo wherein the fungi areselected on the basis of their ability to pass through the livinganimals, whereafter the most stress-tolerant isolates are tested fortheir predacious capacity in a cow dung pat bioassay, and finally

3) the stress selected fungi are optionally tested in a fieldexperiment.

The most preferred fungi which can be selected and isolated in thismanner belong to the genus Duddingtonia and are preferably of thespecies D. flagrans.

Two of the fungal strains selected and isolated by the process of theinvention, Duddingtonia flagrans CI 3 and Duddingtonia flagrans CIII 4,were deposited on Sep. 6, 1991 at DSM--Deutsche Sammlung yonMikroorganismen und Zellkulturen GmbH (German Collection ofMicroorganisms and Cell Cultures) under the respective numbers DSM 6703and DSM 6704.

The present predacious fungi may be administered to the animals in theform of a fungus-containing feed, a fungus-containing feed additive or alick stone to which the fungi have been applied. A supply device in theform of a release bolus can also be inserted into the animal, from whichthe fungus material is released slowly in the alimentary tract.

The effect of the administered fungi is measured in a field experimentas illustrated in FIG. 3 which give average numbers of infectiveOstertagia ostertagi larvae (L₃) transmitted to the grass surroundingcow pats from experimental calves. On day 31 and 55 after the depositionof the cow pats, grass in one half circle (0-30 cm from the edge of thecow pats) was harvested and examined for the number of L3-larvae. k: Cowpats from calves that were not fed with nematode-destroying fungus. a, band c: Cow pats from calved that were fed barley grains with thenematode-destroying fungi (HK II 4)=fungus no. a, (HK II 2 (CIII4))=fungus no. b and (SK II 2 (CI 3))=fungus no. c, respectively.

Fungus no. b and no. c are strains of Duddingtonia flagrans.

The infection of the grass is measured as described in the article"Isolation of Infective Dictyocalus Larvae from Herbage" by R. JessJφrgensen, Veterinary Parasitology 1, 61-67 (1975).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the life cycle of cattle nematode parasites.

FIG. 2 depicts a nematode larva captured by a predacious fungus.

FIG. 3 depicts the effect of the administered fungi in a fieldexperiment. The average number of infective Ostertagia ostertagi larvae(L₃) transmitted to the grass in one-half circle surrounding the cowpats from the experimental and control calves was measured at days 31and 55. k denotes the control group (calves fed no fungi); a denotescalves that were fed grain with fungi strain no. HK II 4; b denotescalves that were fed grain with fungi strain no. SK II 2 (CI 3); cdenotes calves fed with fungi strain no. SK II 2 (CI 3).

DETAILED DESCRIPTION OF THE INVENTION

STRESS SELECTION OF NEMATOPHAGOUS FUNGI

1. In vitro selection of nematophagous fungi for biocontrol of parasiticnematodes in ruminants

Experimental procedure

(A) The demonstration of nematophagous fungi in soil and compost sampleswere made by spreading samples on tetracycline chloride/water agarplates after treatment in sodium hexametaphosphate.

(B) The soil and compost samples were exposed to diluted rumen fluid at39° C. for 24 h. This will be referred to as the primary stressselection step. The addition of buffer (synthetic saliva) to the rumenfluid ensured that pH was kept within the range normally observed in therumen. It has been shown that the situation within test tubes resemblesthe conditions of the rumen when the incubation period was short (J. M.A. Tilley & R. A. Terry, J. Brit. Grassland Society 18, 104-111 (1963);T. Hvelplund & J. Wolstrup, Den Kgl. Veterin.ae butted.r- ogLandbohφjskole's Årsskrift 1974, p. 101-107 (The Royal Danish Veterinaryand Agricultural University Annual)).

(C) The nematophagous fungi, surviving the above primary stressselection step, were further tested in pure culture by exposure to arange of fluids, resembling those of the alimentary tract of cattle.

(D) The most stress-tolerant isolates were tested for their predaciouscapacity in a cow dung pat bioassay. The bioassay elucidated both thecapacity of the isolated fungi to establish in cattle dung and theefficiency of the fungi to capture nematodes in this environment.

A. Demonstration of nematophagous fungi in soil and compost samples

Sources of fungi

Samples of organic farm soil and compost were taken because they usuallyhave a high content of nematophagous fungi (DACKMAN et al., Microbiol.Ecology 3, 89-93 (1987)). Sampling locations are indicated in Table I.

Media for isolation and cultivation of nematophagous fungi

Tetracycline chloride/water agar (TCC-WA) in petri dishes with adiameter of 8 cm were used for the isolation of fungi from soil andcompost. TCC-WA was prepared by mixing sterile filtrated tetracyclinechloride with autoclaved water agar (20 g Difco agar in 1 literdemineralized water) at 45°-50° C. The concentration of tetracyclinechloride in the final media was 0.02% (w/v). The antibiotic was added tosuppress bacterial growth which has previously been shown to makereading of plates difficult. Subsequent isolations and continuoussubcultivation of the fungi were performed using 1:10 corn meal agar(CMA, Difco) plates (B. Nordbring-Hertz, Physiologica Plantarum 23,443-451 (1972)).

Nematodes

Ostertagia ostertagi were obtained from the faeces of calves,experimentally infected with a monoculture of this species. Afterincubation of a faecal culture for 3 weeks at room temperature, thirdstage larvae (L₃) were harvested by a modified Baermann technique. Thefree-living nematode Panagrellus redivivus was cultured axenically in asoy peptone-liver extract medium (B. Nordbring-Hertz, supra). Beforeuse, the larvae of both species were repeatedly washed and resuspendedin sterile water.

                  TABLE I                                                         ______________________________________                                        Soil and compost samples used in primary stress selection                     in diluted rumen fluid                                                        ______________________________________                                        CI    Compost soil containing household organic waste from                          an ecologically managed farm. "Svanholm".                               CII   Compost from a large scale plant. "Dankompost"                                Sengelose (household, agricultural and industrial                             organic waste).                                                         CIII  Compost from a large scale plant in Hong (mixed                               household and garden organic waste).                                    CIV   Compost from private garden containing mainly garden                          waste.                                                                  AI    "Svanholm" agricultural soil used for vegetable                               crops.                                                                  AII   "Askov" agricultural soil. Only fertilizer used                               since 1893 has been pig manure.                                         ______________________________________                                    

Isolation of nematophagous fungi

Samples each of 10 g soil were suspended in 15 ml 0.01% sodiumhexametaphosphate in a 100 ml Erlenmeyer flask for 10 min at roomtemperature. During this period, the flasks were shaken thoroughly.After 5 min rest, 5 replicates of these supernatants (0.5 ml/plate) werespread on TCC-WA plates in a star pattern. Two days later approximately1000-2000 larvae of O. ostertagi and P. redivivus, respectively, wereadded to each of the plates and incubated at room temperature. Duringthe incubation period, nematodes were added to the plates to secure thepresence of living nematodes stimulating the activity of thenematophagous fungi. The plates were inspected under a stereo microscope2-3 times a week for 2 months for growth of nematophagous fungi.

Identification of fungi

The identification of nematophagous fungi was based on the morphology oftrapping structures and conidia (R. C. Cooke & B. E. S. Godfrey,Transactions of the British Mycological Society 47, 61-74 (1964); K.Haard, Mycologia 60, 1140-1159 (1968); N. Jarowaja, Acta Mycologica 6,337-406 (1970) S. Schenck et al., Can. J. Botany 55, 977-985 (1977); K.H. Domsch et al. in Compendium of Soil Fungi vol. 1, 59-64, AcademicPress (1980); C.A.N. van Oorschot, Studies in Mycology 26, 61-96(1985)).

B. Primary stress selection

Test solutions

A buffer solution resembling synthetic saliva was made according to E.I. McDougall, Biochem. J. 43, 99-109 (1948). It was saturated with CO₂at 39° C. and kept at 39° C. until use. Ruminal fluid was collected froma Jersey cow with a ruminal fistula. The fluid was sieved through asieve (mesh size 1 mm) into a 1 liter container and kept at 39° C. Therumen fluid was diluted 1:4 (v/v) with preheated (39° C.) buffersolution (synthetic saliva).

Primary stress selection procedure

For each of the tested soil samples 75 g was added to 225 ml of dilutedrumen fluid in a 300 ml Erlenmeyer flask. The flasks were sealed withairtight rubber stoppers, each supplied with an outlet valve for releaseof gas, and incubated at 39° C. in a shaking waterbath. After incubationfor 24 h the flask rested for 15 min, and the supernatants werecarefully decanted. Five replicates (0.5 ml/plate) of each of thesupernatants were distributed on TCC-WA plates in a star pattern, asdescribed above. The plates were incubated at room temperature.

Stimulation and isolation of nematophagous fungi was carried out asdescribed above (A).

C. Further test of primary stress selected isolates

Test solutions used for in vitro stress selection

Ruminal fluid was collected and stored, as described above, and usedwithout any further dilution. Synthetic saliva was prepared, as alreadymentioned. A pepsin-hydrochloric acid solution simulating abomasal fluidwas made, according to Tilley & Terry (supra), and adjusted to pH 2.5 byadding NaOH.

A 10% trypsin solution (w/v) in demineralized water was prepared, usingDifco trypsin 1:250 and adjusted to pH 7.6. This solution was used as anexample of one of the enzymatic stress factors that would be acting inthe gut.

The solution was heated at 39° C. before use.

All solutions were prepared on the day they were used.

Test procedure for the test of primary stress selected isolates

For each of the primary isolates ten 1:10 CMA petri dishes (diameter 8.5cm) were inoculated with 5×5 mm agar blocks, cut out from the peripheryof a less than 4 weeks old pure culture. Within 5-7 days the fungalmycelium totally covered the petri dishes. After one week the inoculumblocks were removed, and 1 ml preheated (39° C.) test solution forstress selection (described earlier) was added to the plates. Two disheswere used for each solution tested. The mycelium was scraped off the twoplates and the resulting mycelium test media solution was pipetted intoa 10 ml tube containing 5 ml of the test solution. For the rumen fluidtreatment two tubes were established. The tubes were sealed with rubberstoppers, supplied with gas release valves, and incubated in a waterbathat 39° C.

Following 4 h of incubation two samples of 0.5 ml were taken from eachof the tubes, containing buffer solution, pepsin-HCl solution andtrypsin solution. Five samples of 0.5 ml were taken from one of the twotubes with rumen fluid after 24 h of incubation. The material in thesecond tube with rumen fluid was centrifuged (2500 rpm, 10 min) and thesupernatant discarded. 5 ml of pepsin-HCl solution was added to thesediment and, after thorough mixing and adjustment to a pH ofapproximately 2.5 by adding HCl, the material was further incubated for4 h in the waterbath. At the end of this period two samples werecollected and spread on TCC-WA plates. The plates were incubated at roomtemperature and the stimulation and isolation of the nematophagous fungiwas performed, as previously described.

D. Cow dung pat bioassay

Tested fungi

Twelve isolates that survived 24 h treatment in rumen fluid and also 4 htreatment in pepsin-HCl, synthetic saliva and trypsin solutions, wereselected for this test. Two isolates that did not survive the 24 htreatment in rumen fluid (CI 1a and AII 1) were included as negativecontrols.

Cultivation of fungi for bioassay

After treatment with 10% H₂ O₂ for 30 min in a 300 ml Erlenmeyer flaskand washing 3 times in sterile water, 100 g barley grains were mixed 1:1(w:v) with water and auto-claved. The flasks were inoculated with 3-5blocks (5×5 mm) from a less than two weeks old pure culture of theprimary stress selected fungi. The flasks were shaken thoroughly 2 to 3times per week, and the different fungus-grain mixtures were used after2 weeks of cultivation at room temperature (20°-22° C.).

Procedures

Feces from calves infected with a monoculture of O. ostertagi wasdiluted with that from parasite free calves to obtain a concentration of200-400 eggs per gram (EPG), and 125 g was mixed with 40 barley grainswith fungal material. The faeces-barley grain mixture was formed intodome shaped dung pats (approx. 3 cm high). For each fungal isolate, 10of these dung pats were made and placed in plastic boxes (30×21×13 cm)before incubating at 22° C. and 60-80% relative humidity. Following 3and 4 weeks incubation, respectively, 5 dung pats were Baermannized inorder to extract the third stage larvae of O. ostertagi. Dung patscontaining eggs, but not inoculated with fungal material, were incubatedand harvested 5 per treatment at the same time.

The following results were found:

A. Fungi from soil and compost

A total of 55 nematophagous fungal isolates showing a wide variety oftypes were obtained from the plates with soil and compost samples. Sevenspecies of predacious fungi capturing nematodes by sticky branches,constricting rings or sticky nets plus three species of endoparasiticfungi were isolated (Table II).

                  TABLE II                                                        ______________________________________                                        Species of nematophagous fungi isolated from compost and                      soil samples. The type of capture mechanism is indicated                      for the different species of predacious fungi                                 ______________________________________                                        (a) Predacious fungi:                                                             Sticky nets      Constricting rings                                           Duddingtonia flagrans                                                                          Dactynella sp.                                               Arthrobotrys oligospora                                                                        Monacrosporium sp.                                           A. superba       Sticky branches                                              A. amerospora    Monacrosporium cionopagum                                (b) Endoparasitic fungi:                                                          Harposporium anguillulae                                                      Drechmeria coniopora                                                          Verticillium sp.                                                          ______________________________________                                    

B. Primary stress selection

The initial selection step reduced the diversity of nematophagous fungi.

Twenty one isolates belonging to only four species of predacious fungi,all producing sticky nets, survived the primary stress selection indiluted rumen fluid. These isolates could be divided into two groups.The first group consisted of eight isolates showing abundant productionof intercalary chlamydospores (resting spores) and few conidia on short,conidiophores. The chlamydospores were red-brown, thick-walled, eitherspherical with a rough, knobbed appearance or ellipsoid with a smoothsurface. This type of fungi has been identified as belonging to thegenus Duddingtonia with only one species D. flagrans. The second group(thirteen isolates) consisted of three different Arthrobotrys spp. (A.oligospora, A. superba, A. amerospora) producing large amounts ofconidia with one or no septa on branched or unbranched, longconidiophores. The Arthrobotrys spp. also produced chlamydospores, butonly in very limited numbers and only in older cultures (from one to twomonths). The morphological appearance of these chlamydospores differsfrom the type seen in Duddingtonia. The chlamydospores were the smoothtype, ellipsoid to circular, either single or a row of three to fiveintercalary, swollen, thickened cells with usually just one or very fewred-brown pigmented cells in the "chain".

C. Further test of primary stress selected isolates

This qualitative test of the isolates by in vitro stress treatments(Table III) resulted in a further reduction in survivial of isolates,primarily caused by the 24 h treatment in the rumen fluid and rumenfluid with additional pepsin-HCl exposure. In the group of isolatesbelonging to the Arthrobotrys genus, seven out of thirteen, and only oneout of eight of the Duddingtonia isolates, did not show any sign ofgrowth after the rumen treatment. Exposure for 4 h in synthetic saliva,pepsin-HCl solution and trypsin solution did not affect the survival andthe potential to produce nets in the fungi.

                  TABLE III                                                       ______________________________________                                        In vitro stress selection. 13 isolates (A) of nema-                           tophagous fungi belong to the genus Arthrobotrys while the he                 last 8 isolates (B) belong to the genus Duddinggonia. The                     isolate number code refers to the type of sample (Table I                     above). In the table "+" indicates growth and "-"                             indicates no growth.                                                          Treatment*:   1        2     3     4   5**                                    ______________________________________                                        Isolate:                                                                      A:                                                                            CI      1a (control)                                                                            +        -   -     +   +                                    CI      1b        +        -   -     +   +                                    CI      2         +        +   -     +   +                                    CII     1         +        +   -     +   +                                    CII     2         +        +   -     +   +                                    CIII    1a        +        +   -     +   +                                    CIII    2a        +        +   +     +   +                                    AI      1         +        +   -     +   +                                    AII     1 (control)                                                                             +        -   -     +   +                                    AII     2         +        -   -     +   +                                    AII     3         +        -   -     +   +                                    AII     4         +        -   -     +   +                                    AII     5         +        -   -     +   +                                    Isolate:                                                                      B:                                                                            CI      3         +        +   +     +   +                                    CI      4         +        +   -     +   +                                    CI      5         +        -   +     +   +                                    CIII    1b        +        +   -     +   +                                    CIII    2b        +        +   -     +   +                                    CIII    3         +        +   +     +   +                                    CIII    4         +        +   -     +   +                                    CIII    5         +        +   -     +   +                                    ______________________________________                                         *1 synthetic saliva, 4 h: 2 rumen fluid, 24 h; 3 rumen fluid + pepsinHCl      solution, 24 + 4 h; 4 pepsinHCl solution 4 h; 5 trypsin, 4 h.                 **trypsin treatment was only performed once.                             

When the fungi were exposed to the additional effect of simulatedabomasal stress as well as the rumen fluid treatment, only two isolatesof the genus Arthrobotrys and three of the genus Duddingtonia survived.Out of these five isolates two fungi that did not survive the treatmentin rumen fluid for 24 h (treatment 3, table III), unexpectedly survivedthe combined treatment of rumen fluid and pepsin-HCl solution.

                  TABLE IV                                                        ______________________________________                                        The reduction (%) in Ostertagia ostertagi 3rd stage larvae                    after exposure to different isolates of nematophagous fun-                    gi in 125 g dung pats. The dung pats were incubated for 3                     and 4 weeks at approx. 22° C., 60-80% RH. After extraction             by a modified Baermann technique the reduction % was cal-                     culated relative to the controls for the same 2 weeks. The                    isolate number code refers to the type of sample (Table I).                                  %-Reduction in O. ostertagi                                                   L.sub.3 -Larvae                                                               3rd week                                                                             4th week                                                ______________________________________                                        Isolate                                                                       A:                                                                            CI         1a        76       65                                              CI         2         67       85                                              CII        1         74       64                                              CIII       1a        72       86                                              CIII       2a        86       85                                              AI         1         72       73                                              AII        1         74       71                                              B:                                                                            CI         3         91       95                                              CI         4         96       98                                              CIII       1b        86       86                                              CIII       2b        88       86                                              CIII       3         70       80                                              CIII       4         95       94                                              CIII       5         91       94                                              ______________________________________                                         A: Arthrobotrys spp. B: Duddingtonia flagrans                            

D. Cow dung pat bioassay

The addition of fungal material to the dung pats resulted in a reductionof the number of third stage larvae that could be extracted by theBaermann technique (Table IV). The group of Duddingtonia isolatesexhibited a very high reduction capacity (approximately 90%). Withinthis group, only the isolate CIII seemed less efficient. The addition ofthe Arthrobotrys isolates resulted in a more diverse and generally lowerreduction capacity (approximately 75%). By observation of the dung patsduring the incubation period using a stereo microscope with reflectedlight, conidiophores with conidia, sticky nets and the capture of larvaecould be seen on the surface of the dung for species of fungi from bothgenera.

The above results illustrate that the two-step selection technique ofthe invention, simulating the ruminant digestive system, is a usefulmethod for selecting nematophagous fungi with the potential to survivethe stress factors that will be imposed on the organisms during thepassage through a ruminant.

Among the nematophagous fungi isolated after stress selection, theArthrobotrys species were less resistant to stress treatment compared tothe Duddingtonia isolates. The exposure to rumen fluid alone or rumenfluid followed by pepsin-hydrochloric acid treatment appeared to be themain restricting factor. The incubation period for the other threestress treatments were 4 h in contrast to the rumen and rumen plusabomasum simulation that were conducted over a 24 or 24+4 h period,respectively. During this prolonged incubation period, the fungi wereexposed to high temperature in addition to the activity of the rumenfluid microorganisms. In the present study it was not possible todistinguish between the effect of these individual important stressfactors, but the observation that species of Arthrobotrys seem to bevery sensitive to the ruminal stress factors, corresponds with theresults of a preliminary in vitro study (unpublished). In thisexperiment A. oligospora conidia or hyphae survived for two days whenincubated in water, but only for 4 h when exposed to rumen fluid, andfor 4 to 8 h when treated with a pepsin HCl solution, all the treatmentsbeing conducted at 39° C.

The present results could indicate that the production of large numbersof thick-walled spherical chlamydospores of the "rough" type in theDuddingtonia isolates, was responsible for the better survival comparedto the Arthrobotrys spp. exposed to the same stress conditions.

The dung pat bioassay showed that all of the tested fungi had theability to compete and function in this particular environment. Thebioassay also revealed a higher predacious capacity (approx. 90%) in thegroup of fungi belonging to the genus Duddingtonia compared to thecapacity (approx. 75%) found for species of the genus Arthrobotrys.

The differences in survival and predacious capacity between the speciesbelonging to Arthrobotrys and Duddingtonia, clearly indicate thesuperiority of the latter. Duddingtonia spp. seem to be the choice forfuture experiments including oral application of the fungi to ruminantsthroughout an extended period during a grazing season. However, beforesuch a field trial can be performed, it will be necessary to examine thein vivo passage of selected fungi through housed calves.

In vivo passage through calves of nematophagous fungi selected forbiocontrol of parasitic nematodes

The above results have shown that a range of nematophagous fungalisolates are able to survive various in vitro stress treatments,simulating the passage through the gastro-intestinal tract of aruminant. In the following, the in vivo survival and subsequentnematode-reducing capacity of these fungal isolates is tested.

Experimental procedure

Calves were fed for four days with barley grains on which thenematophagous fungi had been cultivated. The daily dose was 200 g (dwt),of which 100 g was given just before the morning fodder (7 a.m.), andthe remaining 100 g just before feeding in the afternoon (3 p.m.). Feceswere collected on day four (morning and afternoon) and day five(morning). After the in vivo passage the fungal material in the faeceswas tested for viability and predacious capacity in a dung pat bioassayand in a faecal culture system. To test the survival of the fungiimmediately after passage, identifiable barley grains were washed out ofthe collected faeces and incubated on water agar plates, supplementedwith antibiotic.

Fungal material

Ten fungal isolates (Table V), proven to survive the above in vitrostress selection, were used in this experiment. The isolates weremaintained on corn meal agar (CMA, Difco) in a dilution of 1:10 (B.Nordbring-Hertz, Nematologica 23, 443-451 (1977)).

Cultivation of fungi

Barley grains were treated with 10% H₂ O₂ for 30 minutes in 400 mlErlenmeyer flask, and washed three times in sterile water. Portions of200 g barley grains were mixed 1:1 (w:v) with water and subsequentlyautoclaved. The flasks were inoculated with three to five blocks (5×5mm) from a less than two weeks old pure culture of the previously stressselected fungi, grown on 1:10 CMA. The flasks were shaken thoroughly twoto three times per week. After two weeks of cultivation at roomtemperature (20°-22° C.), the fungus-grain mixtures were ready to beadministered to experimental calves.

Reisolation of nematophagous fungi

Tetracycline chloride/water agar (TCC-WA) plates (2% Difco agar and0.02% tetracycline chloride) were used for reisolation of the fungi.

Specimens of identifiable barley grains in faeces, from the experimentalcalves, fed fungal material, were washed out of 125 g samples of dung bymeans of a kitchen sieve. Two TCC-WA plates were inoculated with fivegrains each, and inspected under stereoscopic microscope one week laterfor reisolation and identification of the nematophagous fungi.

Identification of fungi

The identification of nematophagous fungi was based on the morphology oftrapping structures and conidia as mentioned above.

Experimental calves

Ten parasite-free Jersey or Friesian Black Pied bullocks (four to sixmonths old), weighing between 118 and 182 kg, were used for the passageexperiment. One calf was used for each of the fungal isolates tested.The experiment was carried out in five sessions, each including two testcalves that were fed fungal material. The calves were kept indoor priorto and during the experiment, and fed with grass pellets and hay.

Parasite material

Faeces, containing eggs of Ostertagia ostertagi, was obtained from twocalves, infected with a monoculture of a Danish strain of this species.In the period when parasite females produce eggs, fresh faeces,containing eggs, was collected for both the dung pat bioassay and thefaecal culture. Feces, without eggs, was obtained from two uninfectedbullocks.

Dung pat bioassay

Faeces from experimental calves, containing fungal material, was diluted1:1 (w/w) with that from calves containing eggs of O. ostertagi. Thenumber of eggs per gram (EPG) in the final mixture was between 200 and300. This mixture, containing both parasite eggs and fungal material,was shaped into 125 g dung pats (approx. 3 cm high, diameter 8 cm). Foreach fungal isolate five dung pats were made and incubated at 22° C. and60-80% relative humidity. After four weeks 0. ostertagi third stagelarvae were extracted from the dung pats, using a modified Baermanntechnique. Dung pats, containing the same number of eggs, but notinoculated with fungal material, were incubated as controls, andharvested five per treatment at the same time.

Faecal cultures

Faecal cultures (10 g faeces samples mixed with 4 g vermiculite and 8 mlH₂₀) were made according to S. A. Henriksen & H. Korsholm, NordiskVeterin.ae butted.rmedicin 35, 429-430 (1983) by using the same faecalmaterial as described for the dung pat bioassay. For each fungal isolatefive cultures were produced. Five cultures containing eggs, but withoutfungal material, served as controls.

The cultures were incubated under the same conditions as the dung pats,and third stage larvae of O. ostertagi were harvested after four weeks.

Statistical analysis

The difference between the number of developed larvae in treatedsamples, containing fungal material, and control samples was tested by aone-tailed rank sum test (Mann-Whitney test).

The following results were found:

Nine out of ten nematophagous fungi were reisolated from the faecesafter passage through the calves (Table V). The reisolated fungi wereall positively identified as the fungi previously given to the calves.

The nematode destroying capacity for eight out of ten fungal isolateswas between 61 and 93% in the dung pat bioassay, and between 76 to 99%in the faecal cultures for seven out of the ten fungi (Table V). Whenthe dung pats and the faecal cultures were inspected under a stereomicroscope, different fungal structures (conidia, trapping organs orchlamydospores ) could be observed directly.

There was a good correlation between the results, obtained from the dungpat bioassay, and the faecal culture technique, except the isolate CI 2.

                                      TABLE V                                     __________________________________________________________________________    Re-isolation and test of reduction capacity of ten fungal                     isolates after passage through calves. Re-isolation of                        fungi, using barley grains washed out of the freshly                          passed faeces, was performed on water agar plates with                        added antibiotic. The reduction capacity was tested in 125                    g dung pats and 10 g faecal cultures incubated for four                       weeks. The difference between used to make inventive                          sample L1 and conventional sample M1. Sample L1 was test samples              and the respective control was tested by a one-tailed rank sum                test (Mann-Whitney Utest). The reduction percentage was                       calculated using the mean values of the fungal isolates                       and respective controls. Labelling of fungal isolates was                     according to Larsen et al., J. Helminthol. 65, 193-200                        (1991).                                                                       Re-isolation                                                                  of fungi    Faecal Cultures                                                   Isolate                                                                             +/-   Median                                                                             Range   Sign.                                                                             Mean                                                                              Red. %                                       __________________________________________________________________________    CIII 2b                                                                             +     277   (211-1868)                                                                           **  771 92                                           CI 2  +     1634  (97-3216)                                                                            **  1463                                                                              85                                           Controls    8592  (6920-15118)                                                                             9630                                             CIII 1b                                                                             +     2414 (1543-2560)                                                                           **  2259                                                                              86                                           CIII 1a                                                                             +     11595                                                                               (6728-18728)                                                                         n.s.                                                                              12295                                                                             26                                           Controls    16161                                                                              (12764-23041)                                                                             16541                                            CIII 3                                                                              +     3788 (2103-6595)                                                                           *   4371                                                                              61                                           CI 3  +     470   (138-1588)                                                                           **  742 93                                           Controls    8678  (4719-20516)                                                                             11040                                            CIII 4                                                                              +     1768  (693-2352)                                                                           **  1505                                                                              85                                           CI 4  -     3000  (1671-15124)                                                                         n.s.                                                                              6675                                                                              32                                           Controls    10874                                                                               (3019-16959)                                                                             9809                                             CIII 2a                                                                             +     725   (592-2607)                                                                           **  1342                                                                              91                                           CIII 5                                                                              +     1710  (180-2639)                                                                           **  1525                                                                              90                                           Controls    14992                                                                               (9208-20493)                                                                             14919                                            CIII 2b                                                                             +     429  (350-493)                                                                             **  409 76                                           CI 2  +     1603 (1275-2137)                                                                           n.s.                                                                              1610                                                                               4                                           Controls    1610 (1507-1921) 1682                                             CIII 1b                                                                             +     247  (223-289)                                                                             **  254 85                                           CIII 1a                                                                             +     1389 (1107-2275)                                                                           n.s.                                                                              1474                                                                              12                                           Controls    1476 (1368-2440) 1674                                             CIII 3                                                                              +     74    (39-102)                                                                             **  77  93                                           CI 3  +     43   (10-71) **  45  96                                           Controls    1269  (182-1800) 1083                                             CIII 4                                                                              +     9     (4-24) **  11  99                                           CI 4  -     887   (304-1443)                                                                           n.s.                                                                              938 11                                           Controls    1031  (760-1466) 1057                                             CIII 2a                                                                             +     30   (21-51) **  33  99                                           CIII 5                                                                              +     31   (28-37) **  28  99                                           Controls    3339 (1757-4280) 3174                                             __________________________________________________________________________     *p <0.05                                                                      **p <0.01                                                                     n.s.: non significant difference                                         

In some previous experiments (not published) it was found that thenematophagous fungus A. oligospora (ATCC 24927) did not survive thepassage through cattle, goats or pigs, either when the fungus was grownand fed to the animals on chopped maize, or as pure fungal material. Thepresent invention has shown that nine out of ten previously stressselected nematophagous fungi are able to survive passage throughruminating calves. Seven out of the ten fungi exhibit a high predaciouscapacity against the free-living larvae of O. ostertagi.

3. Field experiment on biological control of the cattle nematodeparasite Ostertagia ostertagi by feeding host animals with nematophagousfungi

Field experiments as described in the introduction and illustrated inFIG. 3 confirm the ability of stress selected nematophagous fungi tosurvive passage through ruminating calves. Experiments have shown thattwo of the tested fungi, both being Duddingtonia flagrans, are able toreduce the level of infective larvae in the grass by about 70-90%. Onesuch experiment was carried out as follows:

Sixteen calves were divided into two groups with approximately the sametotal body weight and turned out to graze on each of two fields (day 0).Prior to the grazing season each calf was infected orally with 5000infective larvae (L₃) of the gastrointestinal nematode parasiteOstertagia ostertagi. The experimental fields were of the same size withthe same parasitological background, and they contained very fewparasitic nematode larvae in the grass at the time of turn out.

From day 14 until day 67 one group of calves was fed a culture of thenematode-trapping fungus Duddingtonia flagrans (strain SK II 2=CI 3). Onaverage each calf in the treated group was offered 200 g fungal cultureper day in a crib. The fungus was cultured on an autoclaved mixture ofbarley grains and water in the proportion 1:1 (w/v).

Unfortunately it was not raining during the two months feeding period.As parasite L₃ -larvae are mainly spread to the grass during rainyweather, it was not possible to follow the effect of the fungus bymeasuring the number of larvae spread to the grass in this period. Butit was possible to register the fungus as a living component in faecesexcreted by the animals which were offered fungal culture in the dryperiod.

Following the feeding period it started to rain on day 75 and L₃ -larvaewere then spread to the grass.

However, on the fields where calves were fed fungal culture, the grassinfectivity was reduced by 93% on day 82 and by 67% on day 96 comparedto the grass infectivity in the field grazed by the untreated group ofanimals.

Moreover, the treated group of calves started to excrete significantlylower amounts of parasite eggs in their faeces around day 100-110.Compared to the untreated control group the egg excretion was reduced by76%.

The present invention has solved a major problem in the attempt toimplement the use of nematophagous fungi in biological control ofparasitic nematodes in grazing ruminants.

We claim:
 1. A method for reducing the population of nematode parasitesin the faeces of domestic animals susceptible to infection by saidnematode parasites, end thereby reducing the population of saidparasites in pastures, comprising orally administering to the animalspredacious fungi, said fungi being the progeny of fungi having beenpreselected to survive in the fasces of the animal, by being subjectedto a multi-step procedure comprising:(i) In vitro exposure to dilutedruminal fluid at 39° C. for a period of 24 hours; (ii) in vitro exposureto solutions simulating conditions in the alimentary tract of domesticanimals, said solutions comprising or simulating saliva, ruminal fluid,abomasal fluid and a fluid containing an enzyme that acts in the gut andthat subjects said fungi to enzymatic stress; and (iii) in vivo passagethrough the alimentary tract of ruminant mammals, said fungi, after saidprocedure, possessing the ability to reduce the number of parasiticnematodes in a faecal bioassay by at least 50%.
 2. The method forreducing the number of nematode parasites in the fasces of domesticanimals susceptible to infection by said nematode parasites according toclaim 1, wherein the predacious fungi consist of a biologically pureculture of Duddingtonia flagrans CI 3 DSM
 6703. 3. The method forreducing the number of nematode parasites in the fasces of domesticanimals susceptible to infection by said nematode parasites according toclaim 1, wherein the predacious fungi consist of a biologically pureculture of Duddingtonia flagrans CIII 4 DSM
 6704. 4. The methodaccording to claim 1 wherein the predacious fungi are administered inthe form of a feed supplement.
 5. The method according to claim 1wherein the predacious fungi are administered in the form of a lickstone or a release bolus.
 6. A method for the selection of predaciousfungi for use in biological control of parasitic nematodes in domesticanimals, wherein said fungi are subjected to a multi-step selectionprocedure comprising:i) exposing predacious fungi, in vitro, to dilutedrumen fluid at ii) culturing the predacious fungi surviving step (i) toobtain a purified culture of predacious fungi; 39° C. for a period of 24hours; iii) further exposing the purified culture of predacious fungi totest solutions simulating conditions in the alimentary tract ofruminants, in vitro, the test solutions comprising or simulating saliva,ruminal fluid, abomasal fluid and a fluid containing an enzyme that actsin the gut and that subjects said fungi to enzymatic stress; iv) passingthe predacious fungi surviving both steps i) and iii) through thealimentary tract of living ruminants, and subjecting fungi survivingsaid passage to a faecal bioassay to assess the predacious capacity ofthe fungi; and selecting fungi surviving said passage that possess apredacious capacity sufficient to reduce the number of parasiticnematodes in a faecal bioassay by at least 50%.
 7. A method according toclaim 6, further comprising confirming the predacious capacity of thefungi selected in step iv) in dung pats in a field experiment.
 8. Amethod according to claim 6, wherein the exposure to the test solutionsin step iii) comprises contacting the pure culture of fungi with dilutedrumen fluid for 24 hours, followed by contacting the culture for fourhours in each of the following solutions: synthetic saliva; dilutedrumen fluid; pepsin-HCl; and trypsin solution.
 9. The method accordingto claim 6, wherein the selected fungus belongs to the genusDuddingtonia.
 10. The method according to claim 9, wherein the selectedfungus is Duddingtonia flagrans.
 11. A method for biological control ofinfective larvae of parasitic nematodes, comprising administering to ananimal predacious fungi, said fungi being the progeny of fungipreselected by the selection procedure of claim 6 to survive in thefaeces of the animal and possessing the ability to reduce the number ofparasitic nematodes in faeces by at least 50% as measured by a faecalbioassay.
 12. The method for biological control of infective larvae ofparasitic nematodes according to claim 11 wherein the predacious fungiconsist of a biologically pure culture of Duddingtonia flagrans CI 3 DSM6703.
 13. The method for biological control of infective larvae ofparasitic nematodes according to claim 11 wherein the predacious fungiconsist of a biologically pure culture of Duddingtonia flagrans CIII 4DSM
 6704. 14. The method according to claim 11, comprising administeringthe predacious fungi in the form of a fungus-containing feed or a devicereleasing a composition containing said fungi for consumption by saidanimal.
 15. The method according to claim 11 wherein the predaciousfungi are administered to cattle, sheep, goats, pigs or horses.
 16. Themethod according to claim 11 wherein the number of parasitic nematodesin faeces is reduced by more than 60%.
 17. The method according to claim11 wherein the number of parasitic nematodes in faeces is reduced bymore than 75%.
 18. The method according to claim 14 wherein the deviceis a lick stone or a release bolus.
 19. A feed supplement for domesticanimals consisting of predacious fungi, said fungi being the progeny offungi having been preselected by the selection procedure of claim 6 tosurvive in the faeces of the animal and to possess the ability to reducethe number of parasitic nematodes in the faeces by at least 50% asmeasured by a faecal bioassay.
 20. The feed supplement for domesticanimals according to claim 19 wherein the predacious fungi consist of abiologically pure culture of Duddingtonia flagrans CI 3 DSM
 6703. 21.The feed supplement for domestic animals according to claim 19 whereinthe predacious fungi consist of a biologically pure culture ofDuddingtonia flagrans CIII 4 DSM
 6704. 22. A composition for reducingthe population of nematode parasites in the fasces of domestic animalssusceptible to nematode parasitic infection end thereby reducing thepopulation of nematodes in pastures comprising predacious fungi, saidfungi having the property of surviving a multi-step selection procedurecomprising:(i) in vitro exposure to diluted ruminal fluid at 39° C. fora period of 24 hours; and (ii) in vitro exposure to solutions simulatingconditions in the alimentary tract of domestic animals, said solutionscomprising or simulating saliva, ruminal fluid, abomasal fluid and afluid containing an enzyme that acts in the gut and that subjects saidfungi to enzymatic stress; and (iii) in vivo passage through thealimentary tract of ruminant mammals to an extent sufficient to reducethe number of parasitic nematodes in the fasces by at least 50% asmeasured in a faecal bioassay; said composition being adapted to oraladministration to said animals.
 23. The composition for reducing thenumber of nematode parasites in the faeces of domestic animalssusceptible to nematode parasitic infection according to claim 22,wherein the predacious fungi consist of a biologically pure culture ofDuddingtonia flagrans CI 3 DSM
 6703. 24. The composition for reducingthe number of nematode parasites in the faeces of domestic animalssusceptible to nematode parasitic infection according to claim 22,wherein the predacious fungi consist of a biologically pure culture ofDuddingtonia flagrans CIII 4 DSM 6704.